Most strategies to increase marrow production of platelets have focused on use of positive cytokines such as thrombopoietin (TPO). We have recently described an important inhibitory loop that also regulates platelet counts. We found that at least one chemokine that is released by developing megakaryocytes, platelet factor 4 (PF4), acts via a negative paracrine loop to inhibit new megakaryocyte formation and to decrease platelet count. PF4 is normally released by megakaryocytes in amounts sufficient to affect platelet counts in both mice and humans with platelet counts inversely related to PF4 content. Germaine to this application, endogenous PF4 levels affect the degree and duration of thrombocytopenia following both chemo- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Strategies that block this PF4 effect ameliorate the thrombocytopenias. We have also begun to define the molecular basis of this negative paracrine loop. A low density lipoprotein receptor-related receptor (LRP-1) appears to be the target receptor on a late progenitor cell or early maturing megakaryocytes. These observations will now be pursued in 3 specific aims focused on developing useful strategies to ameliorate clinically significant RIT: Specific Aim 1: Examine the efficacy of blocking PF4 on RIT. These studies will examine the ability of blocking polyclonal and monoclonal anti-PF4 antibodies to decrease the severity and duration of RIT in murine models wherein the mice only express human PF4. The efficacy of blocking PF4 in RIT when instituted at various time after the onset of acute radiation syndrome (ARS) and the ability to improve mortality at various radiation exposures will be followed. The ability of other PF4-blocking strategies, such as the infusion of a drug known to compete with PF4 binding, will also be tested. The changes in megakaryocytes and their precursors in the marrow during ARS will also be followed to better understand the underlying biology of the PF4 feedback loop in ARS. Specific Aim 2: Examine the efficacy of blocking LRP-1 on RIT. These studies will extend observations on the role of LRP-1 in PF4's negative paracrine feedback loop in megakaryopoiesis. We plan to study the ability of blocking the LRP-1 receptor as a second approach for limiting the duration and severity of RIT. Specific Aim 3: Combinatorial studies with TPO-based therapies. These studies will take advantage of our observation that eliminating the PF4 negative loop and stimulating the TPO positive loop have an additive effect in improving RIT in ARS. Optimizing combinatorial therapy will be the focus of this specific aim. We believe that we can develop novel strategies based on this negative paracrine loop that can lead to practical therapies to ameliorate the degree and duration of RIT. Further, we anticipate that a strategy that blocks the described negative loop in megakaryopoiesis can be combined with TPO-based therapies to maximize therapeutic outcome after radiation exposure. We believe that we have defined a novel negative paracrine loop that affects megakaryocyte development. Practical strategies based on this regulatory loop should ameliorate the degree and duration of radiation- induced thrombocytopenia and improve overall outcome. Further, we anticipate that this strategy that blocks the described negative loop in megakaryopoiesis can be combined with other therapies, such as thrombopoietin-based therapy, to maximize therapeutic outcome after radiation exposure.